Debaryomyces Hansenii Strains Isolated from Danish Cheese Brines Act As Biocontrol Agents to Inhibit Germination and Growth of Contaminating Molds

Debaryomyces hansenii Strains Isolated From Danish Cheese Brines Act as Biocontrol Agents to Inhibit Germination and Growth of Contaminating Molds

Huang, Chuchu; Zhang, Ling; Johansen, Pernille Greve; Petersen, Mikael Agerlin; Arneborg, Nils; Jespersen, Lene

Published in: Frontiers in Microbiology

DOI: 10.3389/fmicb.2021.662785

Publication date: 2021

Document version Publisher's PDF, also known as Version of record

Document license: CC BY

Citation for published version (APA): Huang, C., Zhang, L., Johansen, P. G., Petersen, M. A., Arneborg, N., & Jespersen, L. (2021). Debaryomyces hansenii Strains Isolated From Danish Cheese Brines Act as Biocontrol Agents to Inhibit Germination and Growth of Contaminating Molds. Frontiers in Microbiology, 12, [662785]. https://doi.org/10.3389/fmicb.2021.662785

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ORIGINAL RESEARCH published: 15 June 2021 doi: 10.3389/fmicb.2021.662785

Debaryomyces hansenii Strains Isolated From Danish Cheese Brines Act as Biocontrol Agents to Inhibit Germination and Growth of Contaminating Molds

Chuchu Huang, Ling Zhang, Pernille Greve Johansen, Mikael Agerlin Petersen, Nils Arneborg and Lene Jespersen*

Department of Food Science, Faculty of Science, University of Copenhagen, Copenhagen, Denmark

The antagonistic activities of native Debaryomyces hansenii strains isolated from Danish Edited by: cheese brines were evaluated against contaminating molds in the dairy industry. Jian Zhao, Determination of chromosome polymorphism by use of pulsed-field gel electrophoresis University of New South Wales, Australia (PFGE) revealed a huge genetic heterogeneity among the D. hansenii strains, which Reviewed by: was reflected in intra-species variation at the phenotypic level. 11 D. hansenii strains Chao-An Long, were tested for their ability to inhibit germination and growth of contaminating molds, Huazhong Agricultural University, China frequently occurring at Danish dairies, i.e., Cladosporium inversicolor, Cladosporium Mark Turner, sinuosum, Fusarium avenaceum, Mucor racemosus, and Penicillium roqueforti. The University of Queensland, Especially the germination of C. inversicolor and P. roqueforti was significantly inhibited Australia by cell-free supernatants of all D. hansenii strains. The underlying factors behind the *Correspondence: Lene Jespersen inhibitory effects of the D. hansenii cell-free supernatants were investigated. Based [email protected] on dynamic headspace sampling followed by gas chromatography-mass spectrometry (DHS-GC-MS), 71 volatile compounds (VOCs) produced by the D. hansenii strains Specialty section: This article was submitted to were identified, including 6 acids, 22 alcohols, 15 aldehydes, 3 benzene derivatives, Food Microbiology, 8 esters, 3 heterocyclic compounds, 12 ketones, and 2 phenols. Among the 71 a section of the journal Frontiers in Microbiology identified VOCs, inhibition of germination of C. inversicolor correlated strongly with Received: 01 February 2021 three VOCs, i.e., 3-methylbutanoic acid, 2-pentanone as well as acetic acid. For Accepted: 20 May 2021 P. roqueforti, two VOCs correlated with inhibition of germination, i.e., acetone and 2- Published: 15 June 2021 phenylethanol, of which the latter also correlated strongly with inhibition of mycelium Citation: growth. Low half-maximal inhibitory concentrations (IC ) were especially observed Huang C, Zhang L, Johansen PG, 50 −5 −4 Petersen MA, Arneborg N and for 3-methylbutanoic acid, i.e., 6.32–9.53 × 10 and 2.00–2.67 × 10 mol/L for Jespersen L (2021) Debaryomyces C. inversicolor and P.roqueforti, respectively. For 2-phenylethanol, a well-known quorum hansenii Strains Isolated From Danish −3 −3 Cheese Brines Act as Biocontrol sensing molecule, the IC50 was 1.99–7.49 × 10 and 1.73–3.45 × 10 mol/L for Agents to Inhibit Germination C. inversicolor and P. roqueforti, respectively. For acetic acid, the IC50 was 1.35– and Growth of Contaminating Molds. 2.47 × 10−3 and 1.19–2.80 × 10−3 mol/L for C. inversicolor and P. roqueforti, Front. Microbiol. 12:662785. doi: 10.3389/fmicb.2021.662785 respectively. Finally, relative weak inhibition was observed for 2-pentanone and acetone.

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Huang et al. D. hansenii Inhibits Contaminating Molds

The current study shows that native strains of D. hansenii isolated from Danish brines have antagonistic effects against speciﬁc contaminating molds and points to the development of D. hansenii strains as bioprotective cultures, targeting cheese brines and cheese surfaces.

Keywords: Debaryomyces hansenii, antagonistic activities, biocontrol, contaminating molds, cheese brine

INTRODUCTION varieties (Gori et al., 2012). Previously, we found that D. hansenii is by far the most predominant yeast species isolated from Danbo Mold contamination is a major problem occurring in food cheese brines reaching ≥ 3.5 log10 CFU/mL (Haastrup et al., processing, which not only leads to severe economic losses and 2018). Moreover, D. hansenii is a highly heterogeneous species food waste but also may influence food safety (Garnier et al., showing phenotypic differences at the strain level. Variations 2017). In the dairy industry, mold contamination can appear among strains include differences in the ability to assimilate throughout the entire cheese production as cheeses come into and ferment different carbon sources, secretion of dissimilar contact with processing equipment and air. Mold growth is lipases and proteases, and diverse preferable growth conditions especially seen during cheese ripening, storage, and distribution (Petersen and Jespersen, 2004). Consequently, strain variations in which results in a reduction of cheese quality including visible antagonistic potential might be expected. Antifungal activities of and invisible defects such as mold colonization, off-flavors, and D. hansenii against contaminating molds are reported for several potential risk of mycotoxin formation (Kure and Skaar, 2019). As foods including dry-cured meat products (Andrade et al., 2014; a consequence, antifungal compounds and potential biocontrol Núñez et al., 2015), fruits (Hernández-Montiel et al., 2010), and agents have received increasing interest to prevent the growth of dairy products (Van Den Tempel and Jakobsen, 2000; Liu and contaminating molds on cheeses (Irlinger and Mounier, 2009). Tsao, 2009; Lessard et al., 2012). In dairy products, D. hansenii Yeasts are considered relevant for biocontrol applications, due is reported to reduce the growth of Penicillium camemberti to their simple cultivation requirements and limited biosafety (Lessard et al., 2012). Further, D. hansenii strains obtained concerns (Freimoser et al., 2019). A number of yeast species from blue mold cheeses are able to weakly inhibit P. roqueforti have been proven to have antagonistic activities against molds, under aerobic conditions (Van Den Tempel and Jakobsen, 2000). though mostly applied in the control of post-harvest diseases of Up to now, several factors have been found to influence the fruits (Bencheqroun et al., 2007; Liu et al., 2013; Grzegorczyk antifungal efficiency of D. hansenii, including water activity et al., 2017; Czarnecka et al., 2019). Within food production, (aw), temperature, nutrient availability (Andrade et al., 2014; antagonistic yeasts have been reported on, e.g., meat and dairy Núñez et al., 2015), and the concentration of molds (Liu and products (Liu and Tsao, 2010; Andrade et al., 2014) but their Tsao, 2009). Further, the production of metabolites varies among application is lacking behind. A number of antifungal volatile D. hansenii strains (Núñez et al., 2015; Grzegorczyk et al., 2017; organic compounds (VOCs) produced by biocontrol yeasts have Hernandez-Montiel et al., 2018). In previous studies, D. hansenii been associated with fungal inhibition, i.e., several alcohols strains have been shown to exhibit varying antifungal actions (2-phenylethanol, ethanol, 2-methyl-1-butanol and 3-methyl-1- (Van Den Tempel and Jakobsen, 2000; Medina-Córdova et al., butanol, 2-methyl-1-propanol, and isoamyl alcohol) (Fialho et al., 2018), however, further in-depth studies are required to explore 2010; Ando et al., 2012; Núñez et al., 2015; Contarino et al., 2019) these actions. We have previously determined the distinctive and esters (ethyl acetate, isoamyl acetate, phenylethyl acetate, growth characteristics and NaCl tolerance of D. hansenii strains isobutyl acetate, 2-phenyl ethyl acetate, and ethyl propionate) isolated from Danish cheese brines (Zhang et al., 2020), however, (Masoud et al., 2005; Fialho et al., 2010; Choinska´ et al., 2020). their potential abilities to inhibit mold contaminants from dairy Although several studies state the production of VOCs involved environments have not, as yet, been explored. in the antifungal activities of biocontrol yeasts, only a few This paper aims to evaluate the antifungal activities of of the aforementioned single VOCs have been investigated in D. hansenii strains isolated from Danish cheese brines against depth. Also other antifungal actions of antagonistic yeasts have different contaminating molds. For this purpose, the effects been reported including the competition for space and nutrients of different D. hansenii strains were examined for their between yeasts and molds (Andrade et al., 2014; Medina-Córdova inhibitory capacity on germination and growth of contaminating et al., 2016) as well as killer toxins playing a role in the defense molds including determination of the half-maximal inhibitory system of yeasts against molds (Santos and Marquina, 2004; concentration (IC50) of single VOCs. Grzegorczyk et al., 2017). A number of yeasts have been detected and isolated from dairy products and dairy environment (Fröhlich-Wyder et al., 2019). MATERIALS AND METHODS Among these, D. hansenii has qualified presumption of safety (QPS) status by the European Food Safety Authority (EFSA) Yeast and Mold Strains, Media, and (Koutsoumanis et al., 2020) making it suitable as a biocontrol Growth Conditions yeast in dairy products. D. hansenii is a halophilic yeasts being All D. hansenii strains used in this study were previously isolated dominating among yeast species associated with most cheese and identified by Haastrup et al.(2018) from three different

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Danish dairies; D. hansenii strains KU-9, KU-10, KU-11, and by the unweighted pair group method using arithmetic average KU-12 were isolated from dairy A (vat1); KU-27, KU-28, KU- linkage (UPGMA method). 29, and KU-30 were isolated from dairy A (vat2); KU-72 was isolated from dairy B; KU-78 and KU-80 were isolated from dairy C (vat1 and vat2, respectively). C. inversicolor, C. sinuosum, Change in pH and Cell Counts of and F. avenaceum were obtained from a Danish dairy and D. hansenii their identification was verified by Deutsche Sammlung von All D. hansenii strains were grown in MYGP broth added Mikroorganismen (DSM, Germany). M. racemosus DSM5266 4% (w/v) NaCl overnight. OD600nm of the overnight cultures (isolated from cheese) and P. roqueforti DSM1079 (isolated were measured using a spectrophotometer (UV 1800, Shimadzu, from cheese) were obtained from DSM. All molds were capable Japan) followed by dilution with fresh media at initial of growing on cheese agar, prepared according to Sørensen concentration of 0.01 ± 0.005 (OD600nm). The cell cultures were ◦ et al.(2011) at 25 ◦C for 7 days (Supplementary Figure 1). grown in MYGP broth added 4% (w/v) NaCl for 72 h at 25 C. Of these, C. inversicolor, C. sinuosum, F. avenaceum, and Measurements of pH were carried out using an electrode (In P. roqueforti reproduce asexually by the formation of conidia, Lab 426, Mettler-Toledo, Glostrup, Denmark) connected to a while M. racemosus reproduce sexually by the formation of pH meter (1120, Mettler-Toledo) and plate counting method spores. In the following, spores will be used when referring to on MYGP agar added 4% (w/v) NaCl was used to monitor the these reproductive cells of the five molds. growth of D. hansenii at the initial and end time point. Yeast strains were propagated at 25◦C in Malt Yeast Glucose Peptone broth added 4% (w/v) NaCl (MYGP added 4% (w/v) Effect of D. hansenii Cell-Free NaCl; per liter, 10 g D (+)-glucose monohydrate (Merck, Darmstadt, Germany), 5 g bactopeptone (BD, Detroit, MI, Supernatants on the Growth of Molds United States), 3 g yeast extract (BD), 3 g malt extract (BD), 40 g Cell-free supernatants were made for each D. hansenii strain in NaCl (Merck) with pH adjusted to 5.3 ± 0.1) or MYGP agar the stationary growth phase (cultured in MYGP broth added 4% ◦ added 4% (w/v) NaCl (adding 20 g bacto agar (BD) to MYGP (w/v) NaCl for 72 h at 25 C) by centrifugation (3000 × g, 10 min) broth added 4% (w/v) NaCl). Mold species were routinely grown followed by filtering the supernatants using 0.22 µm pore size at 25◦C in Malt Extract medium (MEB; per liter; 20 g malt extract filter (Frisenette ApS, Knebel, Denmark). 4 (BD), 10 g D(+)-glucose monohydrate (Merck), 5 g bactopeptone Aliquots of 100 µL of double-strength MEB containing 10 (BD) with pH adjusted to 5.3 ± 0.1) or on malt extract agar (MEA, spores/mL plus 100 µL of the cell-free supernatant collected from adding 20 g bacto agar (BD) to MEB broth components). each D. hansenii strains or 100 µL of Yeast Peptone (per liter, 5 g bactopeptone, 3.0 g yeast extract, 40 g NaCl, pH 5.3) as control were loaded into wells (96 microtiter plates, Corning, New York, Preparation of Spores America). The antifungal activities of cell-free supernatants Molds were cultured on MEA at 25◦C for 7-14 days. Spores against molds were determined by measuring the growth curve were suspended in saline peptone (SPO) solution (per liter; 5 g using oCelloScopeTM Unisensor (Philips BioCell A/S, Denmark) ◦ NaCl (Merck), 0.3 g Na2HPO4·2H2O (Merck), 1 g bactopeptone at 25 C for 48 h. The oCelloScope detection system (objective, (BD) with pH adjusted to 5.3 ± 0.1) containing 0.01% 4×) was described in detail by Fredborg et al.(2013). The image Tween 80 (Sigma-Aldrich, St. Louis, United States) followed by distance was 4.90 µm and the illumination exposure time was filtering through six layers of sterilized gauze to remove hyphal 2 ms. Time-lapse scanning microscopy through a fluid sample fragments. Spore concentrations were estimated by bright field was conducted thereby generating a series of 6 images in each microscopy (Olympus BX40, Japan) using a Neubauer counting well, every 2 h. Growth curves were generated automatically by chamber and adjusted to a final concentration of 104 spores/mL using the segmentation and extraction of surface areas (SESA) using SPO solution. algorithm in UniExplorer (Philips BioCell A/S, Denmark). Germination ratios of spores were analyzed by ImageJ (v1.51g- v1.51n; Fiji package). A spore with a germination tube longer Pulsed-Field Gel Electrophoresis and than the spore itself was considered as a germinated spore. For Cluster Analysis of D. hansenii Strains each image, 90-120 spores were counted and relative germination The DNA preparation of yeast strains and the running condition ratios were calculated as follows: of pulsed-field gel electrophoresis (PFGE) were performed g g according to Petersen and Jespersen(2004). The gels were 1 / 0 × 100% visualized with UV transillumination and photographed t1 t0 (alphaeasefc software, Alpha-InnoTec GmbH, Germany). Estimation of the band size was analyzed using the LabImage where g1 is the number of germinated spores added cell-free 1D, ver.7.1.3 software (Kapelan Bio-Imaging, Germany). The supernatant, t1 is the total number of spores added cell-free cluster analysis was carried out using BioNumerics version 7.1 supernatant, g0 is the number of germinated spores without cell- (Applied Maths, Kortrijk, Belgium). The similarities between free supernatant, t0 is the total number of spores without cell-free profiles of bands were determined using the fraction of shared supernatant. The time points for counting germination ratio were bands (Dice coefficient), and the cluster analysis was calculated chosen according to the method from Trinci(1971).

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Growth rate (µ) values were analyzed using the DMfit were included as controls. The antifungal activities of single software available on the Combase website1 based on the identified VOCs were determined by measuring the growth aforementioned growth curve values and the model proposed by curve using oCelloScopeTM Unisensor at 25◦C for 48 h. Relative Baranyi and Roberts(1994). Relative mycelium growth rate was germination ratios and relative mycelium growth rates were calculated as follows: calculated as described above. In addition, IC50 of single targeted VOCs for each mold was calculated. Long-term impacts of the µ 1 × 100% five single targeted VOCs on the formation of mycelial pellicles µ0 were evaluated by prolonged incubation of the 96-well microtiter plates at 25◦C for 7 days with the concentrations of the five single where µ1 is the growth rate added cell-free supernatant, µ0 is the growth rate without yeast cell-free supernatant. The µ values fit targeted VOCs as listed above. By visual inspection inhibition of with R2 > 0.9 were considered valid data. mycelial pellicle formation was scored.

Determination of Volatile Compounds Quantification of Inhibitory VOCs (VOCs) Based on GC–MS Spectrometry Produced by D. hansenii Strains Debaryomyces hansenii cell-free supernatants were prepared Standard curves based on matrix-spiked samples were performed as described above. VOCs of supernatants were collected in for two acids (acetic acid and 3-methylbutanoic acid), one alcohol a dynamic headspace sampling (DHS) system. Each sample (2-phenylethanol), and two ketones (acetone, and 2-pentanone). contained 20 mL supernatant sample plus 1 mL 4-methyl- The peak area of the quantifier ion was used for estimating 1-pentanol (5 ppm) as the internal standard. The gas flask the concentration of each aforementioned VOCs produced by equipped with a purge head was equilibrated at 37◦C in a D. hansenii strains. water bath with magnetic stirring (200 rpm), and then purged with nitrogen (100 mL/min, 20 min). VOCs were collected Data Analysis by Tenax-TA traps (250 mg, mesh size 60/80, Buchem BV, Data were expressed as mean ± standard deviation and compared Apeldoorn, Netherlands). Then the traps were continually using one-way ANOVA analyzed by SPSS 17.0. Comparison of purged with nitrogen (100 mL/min, 10 min) to remove excess means was performed using Duncan’s multiple range test and water. The dynamic headspace collection was carried out in the statistical significance was applied at the level P < 0.05. duplicates for all samples. VOCs were analyzed using gas IC50 of single VOCs was analyzed by GraphPad Prism 5, while chromatography-mass spectrometry (GC–MS) as described by the Spearman correlation analysis was analyzed by R software Liu et al.(2015) and were identified based on the commercial (“Hmisc” package). database (Wiley275.L, HP product no. G1035A). The identified compounds were confirmed by comparing with retention indices (RI) of authentic reference compounds or the average of retention indices reported in the literature. All identified RESULTS VOCs were semi-quantified as peak areas in the total ion chromatogram (TIC). Debaryomyces hansenii Strains Isolated From Cheese Brines Had Huge Genetic Evaluation of Inhibitory Effects of Single Diversity Targeted VOCs on Molds The chromosomal profiles of the 11 D. hansenii strains isolated Volatile compounds were selected based on correlation between from brines at three different Danish dairies are shown in the relative peak area of each compound produced by D. hansenii Figure 1 and bands sizes are reported in Supplementary Table 1. and the inhibition data (relative germination ratio and relative Among the strains, genetic diversity was evident. The numbers mycelium growth rate obtained from the experiments described of chromosomal bands varied between five and seven with in the section “Effect of D. hansenii Cell-Free Supernatants on sizes from 0.98 to 3.14 Mb. A cluster analysis based on the the Growth of Molds”) through Spearman correlation analysis. chromosomal profiles divided the 11 D. hansenii strains into Those with significant strong correlations (P < 0.05) were four clusters at a similarity level of 60% (Figure 1B). Nine of selected for the subsequent experiments and purchased from the D. hansenii strains had unique profiles, while the two strains Sigma-Aldrich. The interpretation of the rank correlation was in cluster III (KU-11 and KU-28) isolated from two different according to the rules in Akoglu(2018). vats at dairy A (A1 and A2) had identical profiles. D. hansenii Aliquots of 200 µL of MEB containing 104 spores/mL strains (KU-9, KU-10, KU-11, KU-12, KU-27, KU-28, KU-29, supplemented with acetic acid, 3-methylbutanoic acid, acetone, and KU-30) isolated from the two vats at dairy A (A1 and A2) 2-pentanone, and 2-phenylethanol (Sigma-Aldrich, St. Louis, were divided into three different clusters (cluster II, III, and MO, United States) to the final concentrations 100, 10−1, 10−2, IV). Further, strains from dairy C were in cluster I (KU-78 and 10−3, 10−4, 10−5, and 10−6 mol/L were loaded into 96 well KU-80), clearly separated from the remaining dairies. On the plates. Aliquots of 200 µL of MEB containing 104 spores/mL contrary, no clear separation could be observed between dairy A and B as they clustered together in cluster II (KU-9, KU-12, 1http://www.combase.cc/index.php/en/ KU-30, and KU-72), though at low similarity.

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TABLE 1 | Growth of D. hansenii strains and change in pH of MYGP broth added 4% (w/v) NaCl after 72h inoculation at 25◦C.

PFGE Cluster Strain Growth (CFU/mL) after 72h pH after 72 h

I KU-78 3.9 × 107 ± 0.5 × 107a 6.1 ± 0.0f I KU-80 8.3 × 107 ± 0.5 × 107c 5.2 ± 0.1d II KU-12 3.5 × 107 ± 0.9 × 107a 4.5 ± 0.0b II KU-30 2.7 × 107 ± 0.7 × 107a 4.8 ± 0.0c II KU-9 4.0 × 107 ± 0.2 × 107a 4.3 ± 0.2a II KU-72 3.4 × 107 ± 0.4 × 107a 4.5 ± 0.1b III KU-11 3.7 × 107 ± 0.6 × 107a 4.5 ± 0.1b III KU-28 2.6 × 107 ± 0.5 × 107a 4.6 ± 0.1ab IV KU-10 3.9 × 107 ± 0.7 × 107a 6.1 ± 0.1f IV KU-29 4.3 × 107 ± 0.6 × 107a 5.9 ± 0.1e IV KU-27 6.2 × 107 ± 0.6 × 107b 6.1 ± 0.1f

Values in each line marked by different lowercase letters are signiﬁcantly different using one-way ANOVA with Duncan’s test (≥95% conﬁdence, n = 3). Clusters are based on chromosome polymorphism between the stains as determined by PFGE.

growth, acidification of MYGP from pH 5.3 to a minimum of 4.3 was observed for six D. hansenii strains (KU-9, KU-11, KU- 12, KU-28, KU-30, and KU-72). Contrary, deacidification of MYGP from pH 5.3 to a maximum of 6.1 was observed for four D. hansenii strains (KU-10, KU-27, KU-29, and KU-78), whereas one strain (KU-80) did not significantly change the pH during the 72 h growth. Hence, compared to the cluster analysis, the genetic diversity and acidification/deacidification abilities were partly correlated. The acidifying strains were in clusters II and III, while cluster IV comprised three of the four deacidifying strains. Finally, cluster I contained one deacidifying strain (KU-78) and one strain that did not change pH (KU-80), however, rather high genetic differences between the two strains were detected at 62%.

Debaryomyces hansenii Cell-Free Supernatants Affected Germination and Mycelium Growth of Contaminating Molds in a Mold Species Dependent FIGURE 1 | (A) Chromosome polymorphism of D. hansenii strains isolated from Danish cheese brines. Pulsed-field gel electrophoresis (PFGE) was Manner carried out at a 200 switch interval at 150 V for 24 h followed by a 700 s Effects on germination ratios and mycelium growth rates of the switch interval at 100 V for 48 h. Markers: H, Hansenula wingei; S, five molds when exposed to D. hansenii cell-free supernatants Saccharomyces cerevisiae. (B) Dice/UPMGA clustering dendrogram of collected from stationary phase cultures are shown in Table 2. D. hansenii strains. For the germination ratios, the highest significant (P < 0.05) inhibition, among the five molds, was observed for C. inversicolor, with germination ratios below 5% when exposed to cell-free Strain Variations in Acidification and supernatants of all D. hansenii strains. For P. roqueforti, cell-free supernatants of D. hansenii KU-9 and KU-11 showed significant Deacidification Abilities of D. hansenii strong inhibition on the germination ratio to 9.5% and 6.8%, Were Observed respectively, compared to cell-free supernatants of the other Growth and changes in pH of D. hansenii strains grown in D. hansenii strains (13.7-33.8%). Slight inhibition by D. hansenii MYGP added 4% (w/v) NaCl for 72 h at 25◦C are shown in cell-free supernatants was observed on the germination ratio Table 1. Examples of growth curves of D. hansenii strains are of C. sinuosum (82.9%). Contrary, no significant (P > 0.05) shown in Supplementary Figure 2. The initial inocula of the 11 inhibitory effect on the germination ratios of F. avenaceum and D. hansenii strains were 0.7 ± 0.2 × 104 CFU/mL and reached M. racemosus was observed for any of the D. hansenii cell- 4.2 ± 1.7 × 107 CFU/mL after 72 h of growth. Even though free supernatants. all of the 11 D. hansenii strains grew to similar levels there For mycelium growth rates, corresponding to exponential were huge variations in their acidification abilities. After 72 h growth phase of molds, cell-free supernatants of all D. hansenii

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strains induced inhibition of C. inversicolor (69.3%) while slight 4.9 6.6 6.8 4.8 7.4 6.3 1.5 19.6 11.4 13.6 ± ± ± ± ± ± inhibition of P. roqueforti (83.0%) and M. racemosus (86.8%) ± ± ± ± was observed. Mycelium growth rates of C. sinuosum and 1.2 99.3 86.8 83.0 82.9 101.6 20.2 69.3 102.4 101.9 F. avenaceum were not affected by any of the D. hansenii cell- c ab d d bcd ab ab a abcd abc free supernatants. 3.1 4.8 3.1 5.8 2.6 0.4 6.0 4.5 11.0 11.4 ± ± ± ± ± The limited effect of cell-free supernatants collected from ± ± ± ± ± stationary phase cultures of D. hansenii strains were additionally 0.4 33.8 88.0 99.5 97.5 105.2 87.9 77.9 78.0 103.7 confirmed by the growth curves of the molds (Figure 2). a b d bcd cd bc ab abcd

abc Hence, for C. inversicolor and P. roqueforti, the time point ab 4.4 2.9 6.8 3.4 5.9 2.8 4.2 4.1 0.0 of reaching stationary phase was delayed by the D. hansenii 11.9 ± ± ± ± ± ± ± ± ± ± cell-free supernatants, while no inhibitory eﬀect was observed 1.1 98.8 99.0 33.1 85.5 86.9 99.3 86.8 for C. sinuosum, F. avenaceum, and M. racemosus. Moreover, 75.5 100.6 a b d

bc at the end of incubation (i.e., 48 h), the D. hansenii cell-free abc cd bc abc bcd abc 1.1 6.0 3.9 7.9 1.4 3.9

11.0 supernatants did not lead to reduced mycelium growth of any 6.3 2.3 0.9 ± ± ± ± ± ± ± ± ±

± of the ﬁve tested molds. Overall, these results showed that the 1.4 26.1 89.0

67.3 D. hansenii cell-free supernatants predominantly inﬂuenced the 102.7 109.6 63.4 85.0 103.6 101.3 germination phase in a mold species dependent manner, rather ab bc cd c abc abc bc abc bcd bc 2.8 1.8 2.3 than in a D. hansenii strain dependent manner. 6.3 1.0 1.1 1.6 4.0 1.1 11.9 ± ± ± ± ± ± ± ± ± ± 1.9 20.0 88.7 87.5 82.0 100.6 101.4 80.6 100.5 100.0 Debaryomyces hansenii Produced ab a a a ab ab ab a abc bc Antifungal VOCs in a Strain Dependent 2.3 5.9 9.8 4.0 5.8 6.3 0.5 6.0 11.1 0.4 95% conﬁdence, n = 6). Clusters are based on chromosome polymorphism between ± ± ± ± ± ± ± ± ± ± ≥ Manner 6.8 strains 1.9 92.8 92.1 75.8 To elucidate the mechanisms of action involved in the 99.5 99.4 77.0 64.1 74.6 c c antagonistic activities, the VOCs produced by stationary phase b abc bc bc ab a bcd bcd 7.8 3.3

2.9 cultures of the 11 D. hansenii strains were determined. In total, 2.5 3.5 2.2 2.3 0.4 9.2 6.2 ± ± ± ± ± ± ± ± ± ± 71 metabolites were detected by GC-MS and identiﬁed, i.e., D. hansenii 0.4

13.7 6 acids, 22 alcohols, 15 aldehydes, 3 benzene derivatives, 8 90.2 86.7 98.4 111.7 104.5 71.5 83.0 100.5 esters, 3 heterocyclic compounds, 12 ketones, and 2 phenols bc a abc abc abc bcd ab ab d ab

cell-free supernatants as compared to the control. (Supplementary Table 2 lists all detected VOCs). The relative 1.0 6.1 3.7 5.3 4.4 7.7 0.6 2.9 26.0 10.3 ± ± ± ± ± ± ± Table 3

± contents of each metabolite group are shown in . ± ±

4.5 Variations in VOC contents were seen among the D. hansenii 9.5 70.0 98.8 59.7 101.9 102.9 82.0 82.2 104.1

D. hansenii strains. Especially acids, alcohols, benzene derivatives, esters, c c abc bcd ab bc cd abc bcd ab ketones, and phenols were among the produced VOCs. 1.0 4.1 5.5 4.1 2.2 2.7 4.1 8.2 0.8 18.3 ± ± ± ± ± ± Significantly decreased content of aldehydes was detected for all ± ± ± ± D. hansenii strains, except KU-80, while all D. hansenii strains 0.8 20.2 97.3 90.3 86.0 104.0 99.1 78.1 106.8 98.3 had decreased content of heterocyclic compounds. Moreover, b c b abc bcd cd ab abcd abc ab significant contents of acids and ketones were produced by 1.9 4.5 1.3 3.7 4.1 0.5 5.9 6.5 0.4 14.5 ± D. hansenii strains (KU-12 and KU-30). While some D. hansenii ± ± ± ± ± ± ± ± ± strains produced significant contents of benzene derivatives (KU- /h) of molds in presence of 91.2 µ 14.9 86.0 99.6 100.3 83.6 102.9 79.8 9, KU-11, and KU-27), esters (KU-78, KU-9, KU-10, KU-29, and 102.9 bc a a abc abc bcd cd ab KU-27), and phenols (KU-11). Hence, strain-dependent VOCs bcd 2.9 3.6 8.2 0.0 0.8 4.4 5.4 5.9

3.7 proﬁles were obtained from the D. hansenii strains, however, no 11.3 12.6 ± ± ± ± ± ± ± ± ± ± correlation between VOC proﬁles and the genetic clusters from 0.0 96.1 50.9 27.3 96.1

84.9 the PFGE could be observed. 100.8 104.2 83.1 91.6 d ab bc ab a ab bc bc cd To better understand the correlation between the VOCs a 4.8 2.1 1.2 6.9 6.6 0.6 4.6 3.5 0.4 produced by the D. hansenii strains and their antifungal 14.0 ± I I II II II II III III IV IV IV ± ± ± ± ± ± ± ± ± behaviors, Spearman’s rank correlation analysis was applied. 0.4 98.0 74.6 16.2 90.2 85.7 109.5 The results of the correlation and coeﬃcient analysis are 102.6 103.8 56.7 shown in Table 4 and Supplementary Figure 3. Among the 71 VOCs, acetic acid, 3-methylbutanoic acid, and 2-pentanone exhibited a strong negative correlation with germination ratios for C. inversicolor. For P. roqueforti, 2-phenylethanol had a strong C. sinuosum M. racemosus P. roqueforti C. inversicolor C. sinuosum F. avenaceum M. racemosus P. roqueforti Mold species KU-78C. inversicolor KU-80 KU-12 KU-30 KU-9 KU-72 KU-11 KU-28 KU-10 KU-29 KU-27 Average F. avenaceum

Germination ratios (%) and mycelium growth rates ( negative correlation with both germination ratio and mycelium growth rate, while acetone had a strong negative correlation with germination ratio. Thus, these ﬁve VOCs were chosen as targeted TABLE 2 | PFGE cluster Mold growth Germination ratio Values in the same rowthe marked stains by as different determined lowercase by letters PFGE. are signiﬁcantly different using one-way ANOVA with Duncan’s test ( Growth rate single VOCs in the following experiments.

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FIGURE 2 | Effects of cell-free supernatants from D. hansenii strains KU-9, KU-10, KU-11, KU-12, KU-27, KU-28, KU-29, KU-30, KU-72, KU-78 and KU-80 on the growth of ﬁve mold species measured as Normalized SESA units using oCelloScopeTM. (A) Cladosporium sinuosum, (B) Fusarium avenaceum, (C) Mucor racemosus, (D) Cladosporium inversicolor, (E) Penicillium roqueforti. Each spot represents the mean values from three independent experiments, and the error bars represent the standard deviation. (F) Examples of oCelloScopeTM images of growth measurements of C. inversicolor (blue) and P. roqueforti (red) in yeast peptone media (control) or in the presence of cell-free supernatants of D. hansenii strain KU-27 at 24 h. Scale bar: 300 µm.

Targeted Single VOCs Inhibited were 2.47 × 10−3 and 2.80 × 10−3 mol /L for C. inversicolor and Germination, Mycelium Growth, and the P. roqueforti, respectively. 2-pentanone had an inhibitory effect on germination ratios of C. inversicolor and P. roqueforti with an Formation of Mycelial Pellicles of −2 −2 IC50 of 1.06 × 10 and 2.60 × 10 mol/L, respectively. Finally, Contaminating Molds acetone exhibited almost no inhibitory effect on germination The inhibitory effects of targeted single VOCs were examined ratio of C. inversicolor within the experimental period (48 h) successively at different growth stages of C. inversicolor and but weakly inhibited germination ratio of P. roqueforti at IC50 of P. roqueforti, i.e., on germination ratios and mycelium growth 9.21 × 10−2 mol/L. rates within 48 h (Figure 3 and Supplementary Figure 4). Based For mycelium growth rates, gradually increasing on germination ratios and mycelium growth rates in Figure 3, concentrations of 3-methylbutanoic acid, acetic acid, 2- half-maximal inhibitory concentrations (IC50) were calculated phenylethanol, 2-pentanone, and acetone, respectively, were for each growth stage, respectively (Table 5). needed to obtain IC50 for C. inversicolor and P. roqueforti. −5 For the germination ratios, gradually increasing concentration For 3-methylbutanoic acid, IC50 were 6.32 × 10 and of 3-methylbutanoic acid, 2-phenylethanol, acetic acid, 2- 2.67 × 10−4 mol/L for C. inversicolor and P. roqueforti, −3 pentanone, and acetone, respectively, were needed to obtain IC50 respectively. For acetic acid, IC50 were 1.35 × 10 and for C. inversicolor and P. roqueforti. For 3-methylbutanoic acid, 1.19 × 10−3 mol /L for C. inversicolor and P. roqueforti, × −5 × −4 −3 IC50 were obtained at 9.53 10 and 2.00 10 mol/L respectively. For 2-phenylethanol, IC50 were 7.49 × 10 and for C. inversicolor and P. roqueforti, respectively. For 2- 3.45 × 10−3 mol/L for C. inversicolor and P. roqueforti, −3 −3 phenylethanol, IC50 were 1.99 × 10 and 1.73 × 10 mol/L for respectively. Additionally, 2-pentanone showed a weak C. inversicolor and P. roqueforti, respectively. For acetic acid, IC50 inhibitory effect on mycelium growth rate of C. inversicolor

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TABLE 3 | Relative content of volatile organic compounds (VOCs) in each classiﬁcation.

PFGE Sample Acids Alcohols Aldehydes Benzene Esters Heterocyclic Ketones Penols Cluster (n = 6) (n = 22) (n = 15) derivatives (n = 8) compounds (n = 12) (n = 2) (n = 3) (n = 3)

Control 0.06a 81.47a 14.56c 0.01a 1.90ab 0.15e 1.82ab 0.01a I KU-78 0.09ab 87.29b 6.65b 0.05abc 4.80cd 0.08cd 1.02a 0.02ab I KU-80 0.06a 81.28a 14.38c 0.05abc 2.67ab 0.05a 1.29a 0.01a II KU-12 0.14b 89.68bc 3.22ab 0.04abc 3.32abc 0.08cd 3.50c 0.01a II KU-30 0.13b 90.20bc 2.62ab 0.05abc 3.48bc 0.08cd 3.43c 0.02ab II KU-9 0.05a 90.50bc 2.47ab 0.06bc 5.36d 0.09cd 1.45a 0.02ab II KU-72 0.08ab 92.34c 3.09ab 0.03abc 1.69a 0.08cd 2.66bc 0.02ab III KU-11 0.11ab 91.94c 4.53ab 0.06bc 1.89ab 0.10d 1.32a 0.04b III KU-28 0.05a 93.19c 2.45ab 0.05abc 3.21abc 0.06abc 1.00a 0.01a IV KU-10 0.09ab 90.73bc 3.49ab 0.04abc 4.54cd 0.08cd 1.0a 0.01a IV KU-29 0.05a 92.15c 1.58a 0.03abc 5.30d 0.07bc 0.78a 0.03ab IV KU-27 0.08ab 88.97bc 3.77ab 0.07c 5.67d 0.05ab 1.40a 0.01a

The relative percentage of relative peak area of each chemical class of samples, (n) refers to the number of volatile compounds in each class. Values in each classification marked by different lowercase letters are significantly different using one-way ANOVA with Duncan’s test (≥95% confidence, n = 2). Clusters are based on chromosome polymorphism between the stains as determined by PFGE.

TABLE 4 | Spearman’s correlation coefﬁcient analysis between germination ratio (%)/mycelium growth rate (µ/h) and the VOCs detected in the cell-free supernatants of D. hansenii strains.

Mold growth Mold species Volatile compounds R-value (spearman)ab Strength of association

Germination ratio C. inversicolor Acetic acid −0.795 Strong C. inversicolor 3-methylbutanoic acid −0.851 Strong C. inversicolor 2-pentanone −0.731 Strong P. roqueforti Acetone −0.860 Strong P. roqueforti 2-phenylethanol −0.753 Strong Mycelium growth rate P. roqueforti 2-phenylethanol −0.881 Strong

aR = 0 none; −0.3 ≤ R ≤ −0.1 week; −0.6 ≤ R ≤ −0.4 moderate; −0.9 ≤ R ≤ −0.7 strong; −1 perfect. bThe association was considered signiﬁcant (P < 0.05), P-values of all compounds mentioned above were below 0.05.

and P. roqueforti, whereas acetone exhibited no inhibitory effect strains were found to produce estimated concentrations of on mycelium growth rates of the two tested molds. 2-phenylethanol at 10−3 mol/L (data not shown), which was Finally, for confirming the IC50 results as well as for evaluating at the same concentration level as the detected IC50 for both long-term effects of exposure to different concentrations of C. inversicolor and P. roqueforti. The strains were additionally the targeted VOCs, complete inhibition of mycelial pellicle found to produce an estimated concentration of 10−6 mol/L for formation was subsequently scored by visual inspections after 3-methylbutanoic acid and 2-pentanone, and 10−4 mol/L for 7 days incubation (Table 6). As expected, after long-term acetic acid and acetone (data not shown), which were all lower exposure to the targeted VOCs, inhibition of mycelial pellicle than the detected IC50 for both C. invercolor and P. roqueforti for formation confirmed the IC50 results for both C. inversicolor and the respective VOCs. P. roqueforti. Hence, the highest inhibition was obtained for 3- methylbutanoic acid for both C. inversicolor and P. roqueforti, for which the concentrations were 10−3 and 10−2 mol/L, DISCUSSION respectively. The concentrations of 2-phenylethanol and acetic acid for preventing the formation of mycelial pellicles were 10−2 In this study, we evaluated D. hansenii strains isolated from and 10−1 mol/L for C. inversicolor and P. roqueforti, respectively. Danish cheese brines for their antifungal activities against dairy Furthermore, the concentration of 2-pentanone was 1 mol/L for contaminating molds. Our PFGE result confirmed the genetic C. inversicolor, while no inhibition on mycelial pellicle formation heterogeneity of D. hansenii as previously reported (Petersen was observed for P. roqueforti after 7 days. Finally, acetone et al., 2002; Petersen and Jespersen, 2004). In Petersen et al. did not inhibit mycelial pellicle formation of either of the two (2002), chromosome polymorphism among D. hansenii strains molds after 7 days. could be linked to different pH and NaCl tolerances. The Among the targeted VOCs, estimated concentrations variations in acidification or deacidification capabilities among produced by the D. hansenii strains were calculated, when the D. hansenii strains, found in the present study, could to grown under the experimental conditions. The D. hansenii some extend be linked to the genetic variance observed. The

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FIGURE 3 | Inhibitory effects of targeted single VOCs on germination ratios (%) of C. inversicolor (A) and P. roqueforti (C) and mycelium growth rates (µ/h) of C. inversicolor (B) and P. roqueforti (D) at different concentrations (100, 10−1, 10−2, 10−3, 10−4, 10−5, and 10−6 mol/L). Each spot represents the mean value from three independent experiments, and the error bars represent the standard deviations.

TABLE 5 | IC50 of different VOCs for inhibiting germination and mycelium growth Strain-dependent VOC profiles have previously been observed of P. roqueforti and C. inversicolor. among D. hansenii strains differing in M13 minisatellites, isolated Mold growth Volatile compounds C. inversicolor P. roqueforti from traditional ewe’s and goat’s cheese (Padilla et al., 2014b). Among the five contaminating molds, P. roqueforti is, besides

IC50 (mol/L) being a contaminant on surface ripened cheeses, a well known starter culture used in the production of blue-veined cheeses. Germination Acetic acid 2.47 × 10−3 2.80 × 10−3 ratio Mucor spp. are well known on a variety of mold-ripened cheeses, 3-methylbutanoic acid 9.53 × 10−5 2.00 × 10−4 especially from raw milk hard cheeses such as Saint Nectaire Acetone >1 9.21 × 10−2 and Tomme de Savoie (Fox et al., 2004), and M. racemosus 2-pentanone 1.06 × 10−2 2.60 × 10−2 has especially been isolated from French cheeses (Hermet et al., 2-phenylethanol 1.99 × 10−3 1.73 × 10−3 2015). Further, F. avenaceum has predominantly been reported Mycelium Acetic acid 1.35 × 10−3 1.19 × 10−3 as a mycotoxin producing pathogen on cereals, especially growth rate pronounced under cool and wet or humid conditions (Uhlig 3-methylbutanoic acid 6.32 × 10−5 2.67 × 10−4 et al., 2007), however, it has also been isolated as a contaminating Acetone >1 >1 mold on Italian cheese (Montagna et al., 2004). Likewise, 2-pentanone 9.45 × 10−1 9.51 × 10−1 F. avenaceum was isolated at Danish dairies and identified to 2-phenylethanol 7.49 × 10−3 3.45 × 10−3 the species level in the present study. Finally, Cladosporium spp. have been isolated from traditional Italian cheese (De Santi et al., 2010). Originally isolated at Danish dairies, C. inversicolor and pH differences of the broth media (MYGP added with 4% (w/v) C. sinuosum were identified to species level, for the purpose NaCl) might be related to D. hansenii producing or assimilating of this study. However, C. inversicolor and C. sinuosum are organic acids, proteolytic activity, production of ammonia, cell so far mostly reported from plant materials (Bensch et al., lysis, as well as several other factors (Gustafsson, 1979; Gancedo 2015). Altogether, this indicates a huge species diversity among and Serrano, 1989; Freitas et al., 1999; Flores et al., 2004; Gori contaminating molds in dairy environments, far beyond what is et al., 2007, 2012). Contrary, the VOCs profiles produced by the usually investigated. D. hansenii strains, in the present study, could not be linked to the In the present study, the D. hansenii cell-free supernatants specific chromosomal clusters as observed by PFGE, even though inhibited spore (conidia) germination and had only minor strain dependent differences in VOC production were observed. inhibitory effect on mold growth, indicating a fungistatic

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effect, which might be associated with the morphology of the lowest IC50 against germination and mycelium growth the spores or conidia of the five molds. Interestingly, the of C. inversicolor and P. roqueforti at 10−5 and 10−4 mol/L, two Cladosporium spp., i.e., C. inversicolor and C. sinuosum respectively. The sensory threshold for 3-methylbutanoic acid is responded very different to the D. hansenii cell-free supernatants. reported to 120-700 ppb (1.17-6.85 × 10−6 mol/L) and at higher Germination and mycelium growth of C. inversicolor were concentrations, this branched-chain fatty acid has been described significantly inhibited by cell-free supernatants of the D. hansenii to contribute with unpleasant odors as floor cloth, feet, and strains, whereas we only observed a slight inhibition of sweaty flavors in surface ripened cheeses from France (Lecanu germination for C. sinuosum and no effect on mycelium et al., 2002). The estimated concentration of 3-methylbutanoic growth. The Cladosporium genus represents one of the acid (10−6 mol/L) detected in the present study was therefore larges genera of dematiaceous hyphomycetes and comprises within the sensory thresholds but lower than the IC50 values several complexes, which are differentiated morphologically mentioned above. Even so, the potential use of D. hansenii based on especially conidia morphology. C. inversicolor is strains producing 3-methylbutanoic acid as a biocontrol agent in the Cladosporium cladosporioides complex and contains is still interesting. Higher levels of 3-methylbutanoic acid might numerous conidia in long chains of up to eight conidia potentially be obtained under different processing conditions, as with walls typically being unthickened. C. sinuosum is in reported by Durá et al.(2004) where NaCl and lactate increased the Cladosporium herbarum complex and contains solitary the levels of 3-methylbutanoic acid produced by D. hansenii, and conidia or short chains of up to four conidia which appear detailed analyses still need to be carried out in cheese under to be thick walled due to surface ornamentation (Bensch relevant maturation conditions. et al., 2015). These differences might explain some of the The VOC 2-phenylethanol is reported as a common variance in the susceptibility observed between the two inhibitory compound produced by several biocontrol yeasts Cladosporium species. including Debaryomyces nepalensis, Wickerhamomyces anomalus, Among the 71 VOCs produced by D. hansenii, five Metschnikowia pulcherrima, Saccharomyces cerevisiae, and Pichia targeted VOCs (acetic acid, 3-methylbutanoic acid, acetone, manshurica (formerly, Pichia galeiformis)(Zhou et al., 2018; 2-phenylethanol, and 2-pentanone) had a strong association Contarino et al., 2019; Chen et al., 2020). In this study, we found with inhibition of germination and/or mycelium growth of a strong inhibitory effect of 2-phenylethanol on germination C. inversicolor and P. roqueforti and were selected for detailed and mycelium growth of C. inversicolor and P. roqueforti at analyses. Accordingly, D. hansenii has been shown to produce 10−3 mol/L. Accordingly, we have previously found that 2- acetic acid, acetone, 3-methylbutanoic acid, 2-pentanone, and phenylethanol completely impaired conidia germination, hyphal 2-phenylethanol in cheese-like medium (Padilla et al., 2014a). membrane integrity, and growth of another two food spoilage Moreover, D. hansenii increased the amount of 2-pentanone molds, i.e., Penicillium expansum and Penicillium nordicum in feta cheese (Bintsis and Robinson, 2004). In our study, 3- at 15 × 10−3 mol/L (Huang et al., 2020). The concentration methylbutanoic acid had the strongest inhibitory effect and of 2-phenylethanol produced by D. hansenii strains in the

TABLE 6 | Inhibitory effects of ﬁve VOCs on the formation of mycelial pellicles of molds after 7 days.

Mold species Concentration (mol/L) VOCs

Acetic acid 3-methylbutanoic acid Acetone 2-pentanone 2-phenylethanol

C. inversicolor 100 +++ +++ − +++ +++ 10−1 +++ +++ − − +++ 10−2 ++ +++ − − + 10−3 − ++ − − − 10−4 − − − − − 10−5 − − − − − 10−6 − − − − − P. roqueforti 100 +++ +++ − − +++ 10−1 +++ +++ − − +++ 10−2 − +++ − − − 10−3 − − − − − 10−4 − − − − − 10−5 − − − − − 10−6 − − − − −

+++ no mycelial pellicles formed. ++ less than or equal to 3 out of 6 replicates forming mycelial pellicles. + less than or equal to 5 out of 6 of replicates forming mycelial pellicles. − no inhibition.

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present study was approximately 10−3 mol/L and therefore relevant. Interestingly, D. hansenii isolated from dry-cured meat −3 comparable with the IC50 (10 mol/L level), thus highlighting products has been reported to inhibit toxigenic penicillia in the potential application of the D. hansenii strains for biological co-culture assays on solid media, which could not be fully production of 2-phenylethanol as an antifungal agent. Moreover, reproduced by cell-free supernatants or mouth-to-mouth assays. 2-phenylethanol is a well-known quorum sensing molecule This indicates that efficient mold inhibition relied on additive produced by D. hansenii, and is found to upregulate traits or synergistic effects of the yeast inhibition factors such as involved in adhesion and sliding motility of D. hansenii competition for nutrients and space as well as production of (Gori et al., 2011), increasing the colonization probability soluble compounds or VOCs (Núñez et al., 2015). Similarly, P. on cheese surfaces (Mortensen et al., 2005; Gori et al., manshurica has been shown to colonize and amplify quickly in 2011), which in turn could contribute to the quality of citrus wounds and further to produce eight VOCs, including the final product. acetic acid and 2-phenylethanol during growth, which inhibited Acetic acid is a common antimicrobial compound and growth of Penicillium digitatum, causing citrus green mold its antifungal activity is well investigated (Cabo et al., 2002; (Chen et al., 2020). Fernandez et al., 2017; Guimarães et al., 2018; Sadiq et al., 2019). In conclusion, genetic differences of D. hansenii together −3 In this study, the IC50 for acetic acid was found at 10 mol/L with diverse acidifying or deacidifying capability were confirmed. against C. inversicolor and P. roqueforti. Quattrini et al.(2019) Cell-free supernatants of D. hansenii strains exhibited a reported the minimum inhibitory concentration (MIC) of acetic strong inhibitory effect on C. inversicolor and P. roqueforti. acid for P. roqueforti growth to 2.5 ± 0.6 × 10−2 mol/L Using DHS-GC-MS, 71 VOCs produced by D. hansenii after 5 days, which is somewhat lower than the concentration strains were identified. Among the VOCs, strong correlation of acetic acid for preventing mycelial pellicle formation of between inhibition of germination of C. inversicolor and P. roqueforti (approximately 10−1 mol/L) after 7 days, in P. roqueforti were obtained for five VOCs (acetic acid, acetone, the present study. In Quattrini et al.(2019), MIC was 3-methylbutanoic acid, 2-pentanone, and 2-phenylethanol), determined after 5 days in conditions mimicking sourdough while 2-phenylethanol also correlated strongly with inhibition using modified MRS, while in the present study inhibition of mycelium growth of P. roqueforti. 3-methylbutanoic acid was scored after 7 days in MEB, a medium for optimal showed the strongest inhibitory effects on germination and mold growth. These differences could explain the different mycelium growth of the two mold species, i.e., IC50 of − − concentrations determined. 10 5 and 10 4 mol/L, respectively. 2-phenylethanol and acetic In soft cheeses, 2-pentanone has been detected in Camembert, acid also exhibited strong inhibitory effects on germination −3 Vacherin, Limburger, Trappist, Gorgonzola and blue cheese and mycelium growth at IC50 of 10 mol/L. Relative above the odor threshold of 0.5-61 ppm contributing fruity, weak inhibitory effects of 2-pentanone and acetone were acetone, sweet, and ethereal odor (Sablé and Cottenceau, obtained in this study. The results from the current study 1999). In the current study, 2-pentanone showed a weak point to an additive and possibly synergistic effect of the inhibitory effect on the germination and mycelium growth antifungal compounds produced by D. hansenii, which needs of C. inversicolor and P. roqueforti. Further, the estimated to be further studied. From an overall perspective, it can concentration of 2-pentanone produced by D. hansenii in be concluded that some native D. hansenii strains in the this study, was lower than the detected IC50 but within dairy manufacturing environment have a so far neglected role the ranges of odor threshold reported in previous studies in the natural preservation of cheeses and are eligible for (Sablé and Cottenceau, 1999). biocontrol of contaminating molds in cheese production. The The results of the present study indicate that the antifungal results additionally point to the coming use of halophilic activity of D. hansenii is complex and likely results from a D. hansenii strains as biocontrol agents to be added as synergistic and or additive effect of several inhibitory VOCs, bioprotective cultures in the dairy industry, e.g., as an additive rather than the effect of single compounds. Similarly, growth to cheese brines. inhibition of postharvest pathogenic molds in packaged fresh fruit by the proven biocontrol yeasts W. anomalus BS91 and M. pulcherrima MPR3 has been suggested to arise from a synergistic effect of VOCs and CO2 (Contarino et al., 2019). DATA AVAILABILITY STATEMENT Moreover, inhibition of Fusarium spp. growth in the presence of cell-free supernatants of Lactiplantibacillus plantarum (formerly, The original contributions presented in the study are included Lactobacillus plantarum) has been shown not to arise alone in the article/Supplementary Material, further inquiries can be from the lactic acid being produced by the lactic acid bacteria directed to the corresponding author/s. (Laitila et al., 2002). Further, additional mechanisms, not investigated in the current study, might also add to the combined antifungal activity of D. hansenii, including non- volatile compounds like killer toxins (Banjara et al., 2016), AUTHOR CONTRIBUTIONS and competition for nutrients and space (Spadaro and Droby, 2016). As nutrients and space are generally limited in cheese CH, LZ, PJ, NA, and LJ conceived and designed the study. processing (Irlinger and Mounier, 2009), this could be especially MP contributed to the GC-MS analyses. CH performed the

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experiments and drafted the manuscript. PJ, NA, and LJ revised by the Chinese Scholarship Council (CSC201706350030 and the manuscript. All authors contributed to the article and CSC201706350054). approved the submitted version.

FUNDING SUPPLEMENTARY MATERIAL

This work was conducted as a part of the Danish InBrine project. The Supplementary Material for this article can be found The authors are grateful to the Danish Dairy Board (MFF) online at: https://www.frontiersin.org/articles/10.3389/fmicb. for funding of the research. Additional funding was obtained 2021.662785/full#supplementary-material

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cultures for production of Danablu. Int. Dairy J. 10, 263–270. doi: 10.1016/ Conflict of Interest: The authors declare that the research was conducted in the S0958-6946(00)00053-4 absence of any commercial or financial relationships that could be construed as a Zhang, L., Huang, C., Malskær, A. H., Jespersen, L., Arneborg, N., and potential conflict of interest. Johansen, P. G. (2020). The effects of NaCl and temperature on growth and survival of yeast strains isolated from Danish cheese Copyright © 2021 Huang, Zhang, Johansen, Petersen, Arneborg and Jespersen. This brines. Curr. Microbiol. 77, 3377–3384. doi: 10.1007/s00284-020- is an open-access article distributed under the terms of the Creative Commons 02185-y Attribution License (CC BY). The use, distribution or reproduction in other forums Zhou, Y., Li, W., Zeng, J., and Shao, Y. (2018). Mechanisms of action of the is permitted, provided the original author(s) and the copyright owner(s) are credited yeast Debaryomyces nepalensis for control of the pathogen Colletotrichum and that the original publication in this journal is cited, in accordance with accepted gloeosporioides in mango fruit. Biol. Control 123, 111–119. doi: 10.1016/j. academic practice. No use, distribution or reproduction is permitted which does not biocontrol.2018.05.014 comply with these terms.

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